Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine), an analog of creatine and a substrate for creatine kinase (EC 22.214.171.124), inhibits the stimulated motility of tumor cells which possess creatine kinase. A2058-055 human melanoma cells, transfected with a creatine kinase gene, showed an 80-90% reduction in chemotactic response to type IV collagen when incubated overnight in the presence of 10 mM cyclocreatine (p < 0.0001 for n = 8 experiments). This inhibitory effect of cyclocreatine can be partially reversed by addition of creatine to the overnight cell treatment. Non-transfected cells, with very low levels of creatine kinase, were not significantly inhibited. Further experiments utilizing type IV collagen as attractant demonstrated that cyclocreatine inhibited the chemokinetic (91%) and the haptotactic (73%) responses and the in vitro invasion of A2058-055 cells through Matrigel-coated membranes (88%). In addition, motility stimulation of A2058-055 cells by either autotaxin or fibronectin was markedly inhibited by cyclocreatine. DU-145 prostatic tumor cells, which express endogenous creatine kinase, also have a reduced motility response to either autotaxin or epidermal growth factor induced motility in the presence of cyclocreatine.